Niridazole Biodegradable Inserts for Local Long-Term Treatment of Periodontitis: Possible New Life for an Orphan Drug

Abstract

Periodontal pocket inserts of niridazole (NZ) made with Resomer® (grades RG 503H and RG858, designated as RH and RG, respectively) were studied. Various formulation variables were evaluated to obtain a biodegradable delivery systems showing device degradation and drug depletion parallel to each other in vitro. Drug release from the prepared inserts was evaluated using a static dissolution setup (for 1 month). Incorporation of 3 parts of RG in 1 part of RH inserts caused a 50% decrease in the initial release rate. The RH-NZ inserts showed a spurt in release around the 10th day of the study, which coincided with the decrease in device weight, suggesting onset of device degradation. Pilot-scale clinical trials in 12 patients indicated improvements in clinical indices from the baseline values. The average pocket depth was reduced significantly (α = 0.05) from 6.34 ± 1.86 mm at baseline to 5.94 ± 0.28 mm after 28 days of treatment.

Keywords :

• Biodegradable
• Niridazole
• Periodontitis
• Resomer®
• RG 503H
• RG858

Periodontal diseases are inflammatory conditions of predominantly microbiological origin affecting the structural organs supporting the teeth, initially involving the gingival tissue and slowly progressing to the periodontium. If treatment is not initiated, irreversible loss of tooth support structures result in tooth loss (Becker, Berg, and Becker 1979; Buckely and Crowley 1984; Lindhe, Haffajee, and Socransky 1983).

Conventional treatment of periodontitis includes periodic mechanical debridement of plaque from tooth surfaces and repeated topical or systemic administration of antibacterial agents (Hellden, Listergarten, and Lindhe 1979; Joyston-Bedral 1987; Lindhe and Nyman 1984; Ramfjord 1987; Vander-Oudera 1991). The effectiveness of the conventional treatment is, however, limited by the lack of accessibility of the periodontopathic organisms inside the periodontal pocket (Medlicott, Tucker, and Holborow 1994). Systemic administration of antibacterial agents have achieved therapeutic concentrations at the site of infection (Gordon et al. 1981; Van Oosten, Notten, and Mike 1986; Higashi et al. 1989; Britt and Pohlod 1986) for short periods of time after a single dose, and repeated dosing for longer periods leads to many side effects (Slots and Rams 1990).

To overcome the shortcomings of the conventional modes, prolonged release intrapocket delivery systems have been developed (Arrona et al. 1998; Ciancio 1999; Kinnane and Radvan 1999; Somayaji et al. 1998; Garret et al. 1990). The greatest advantage of intrapocket delivery systems is that treatment does not depend heavily on patient compliance. Also, the amount of drug required to achieve effective concentration in gingival crevicular fluid (GCF) is considerably less than a systemic regimen. Biodegradable devices offer the advantage over nonbiodegradable devices in terms of patient compliance as device removal is not required.

Niridazole (NZ), a substituted nitrothiazole derivative, has been successfully used in schistosomiasis (Mahmoud 1977) and other helminthic infections. Following the discovery of praziquantel, a safer alternative, NZ was used clinically as a second line drug (Andraus, Thomas, and Szubert 1983) and its use was slowly phased out following incidents of high dose-related side effects. In view of the almost total lack of use in other human diseases, Wade and Addy (1987) suggested that NZ is worthy of evaluation in periodontal patients. Further, the low minimum inhibitory concentration (0.25 mg/l) of NZ for periodontopathogens (Hof and Stroder 1986; Hof and Sticht-Groh 1984; Hof et al. 1985) requires very low doses of NZ for intrapocket delivery; consequently almost no systemic side effects are expected.

Devices based on polymers of lactic and glycolic acids (Resomers®) are widely used in a number of biomedical applications (Lewis 1990). The biodegradability and nontoxic nature of the hydrolysis product makes it an excellent material of choice for applications that require either insertion or implantation of the device within the biological milieu.

The objective of our study was to develop film-type inserts of NZ using Resomer® for long-term treatment of periodontal disease. The procedure for the preparation of the films was optimized. The properties of the films were studied and in vitro characteristics and pilot scale clinical trials of the inserts in patients suffering from periodontal disease were investigated. The possibility of NZ reaching the systemic circulation was ruled out by HPLC estimation of NZ in the plasma of volunteers, and the safety of NZ in this mode of treatment was confirmed.

MATERIALS AND METHODS

Niridazole was obtained as gift sample from Egis Pharmaceuticals Ltd. (Hungary). PLGA grades RH and RG (Resomer® RG 503H and RG858) were purchased from Boehringer Ingelheim, KG (Germany). All other reagents used were of analytical and HPLC grade.

Preparation of Inserts

RH and RG were used alone or in combinations as shown in Table 2. The required ratios of the resomers were weighed and added to acetone in a covered beaker under stirring. The mixture was stirred for 4–6 hr untill a clear solution was obtained. NZ was sieved through #300 and suspended in acetone to give a slurry. The slurry was added to the polymer solution through a stainless steel #300 mesh under stirring. Plasticizer (propylene glycol) was then added to the drug-polymer mixture. The resultant polymeric mixture was cast on Scochpak® liner and dried at room temperature under an inverted funnel for 30 hr. Upon drying, the films were wrapped in butter paper and stored in amber-colored glass vials in a desiccator until further use. The films were subdivided into inserts (2 × 6 mm) by punching out.

TABLE 2 Composition of the prepared inserts

Batch code	RH:RG ratio	NZ (mg/insert)	Polymer content (mg/cm^2)
RN1	1:0	0.5	10
RN2	1:0	0.5	50
RN3	1:0	0.5	60
RN4	1:0	1.0	50
RN5	1:3	0.5	60
RN6	3:1	0.5	60
RN7	1:1	0.5	60
RN8	0:1	0.5	10
RN9	0:1	0.5	50
RN10	0:1	0.5	60
RN11	0:1	1.0	50

RH = Resome 503H, RG = Resomer 858, NZ = Niridazole.

Evaluation of Inserts

Thickness, Weight, and Drug Content Uniformity

The thickness of the inserts was measured at five different randomly selected spots with a screw gauge. For uniformity of weight, 10 inserts from each batch were weighed individually and their average determined. For determination of uniformity of drug content, 6 inserts from each batch were weighed individually and dissolved in 50 ml of McIlvaine's buffer pH 6.6. The resultant solution was filtered through a G-2 glass filter. An aliquot of the filtrate was suitably diluted and analyzed for NZ content at 365 nm (Shimadzu, UV-1601, Japan).

In Vitro Release Studies

Weighed inserts were placed in a stainless steel wire mesh holder of dimensions 2 × 4 × 6 mm and suspended in amber-colored vials containing 10 ml of McIlvaine's buffer pH 6.6 as the dissolution medium. The vials were stoppered and placed on a vial holder fitted in a water bath thermostated at 37 ± 1°C. At predetermined time intervals the dissolution medium was completely withdrawn and replaced with a fresh 10 ml portion of the prewarmed buffer to ensure sink conditions. The samples were analyzed for NZ content at 365 nm, after appropriate dilution.

Water Uptake Measurement

Swelling rate was determined by immersing a previously weighed insert in stainless USP dissolution basket (preweighed), in 50 ml of McIlvaine's buffer pH 6.6 at 37 ± 1°C. At predetermined time intervals the basket containing insert was removed, wiped with tissue paper to remove the surface buffer, weighed, and finally replaced in buffer. The relative water uptake was calculated using the relationship as given by Bottenberg et al. (1992). Relative water uptake = (SW₂ - SW₁)/SW₀. Where SW₁ is weight of the USP basket alone, SW₂ is weight of swollen device and USP basket, and SW₀ is initial weight of the insert.

Pilot-Scale Clinical Trial of Drug-Loaded Inserts

Approval of the ethical committee of the institute (Banaras Hindu University, India) was obtained prior to the commencement of study. The design of the trial followed a single-blind parallel group with matched pair design. Twelve patients (18–55 years of age, either sex), constituting 24 sites each in treatment and control, were selected from the outpatient department at Department of Dentistry, Institute of Medical Sciences, Banaras Hindu University. The patients were quizzed about their current ailments, previous treatment if any, general health status, and so on, as per a questionnaire. From the data acquired, patients with at least 4 periodontal pockets with depth equal to or greater than to 6 mm, not adjacent to each other and are of normal general health statue were included in the study. Pregnant or lactating women and patients with a history of antibiotic and/or periodontal therapy within the last 6 months were excluded from the clinical study. The selected patients were briefed about the treatment types (full mouth scaling and periodontal therapy), treatment duration, number of follow-up visits, and the benefits and risks involved. After obtaining written consent from the patients to participate in the trial, oral hygiene instructions (OHI) were given.

Prescaling, depending upon the severity of calculus and plaque and accessibility of the subgingival sulcus, was carried out in 2–3 sittings at intervals of 7 days. The maximum time interval for complete supragingival scaling between initiation of prescaling and Day 0 (baseline) was 21 days and the minimum was 14 days. Full mouth scaling was carried out using subgingival and supragingival scales.

The sites for the trial were randomly selected and 4 sites per patient were allotted—2 each to control group (scaling and OHI only) and to the treatment group (scaling, OHI, and placement of drug-loaded insert into the periodontal pocket). The clinical parameters like gingival index (GI), bleeding index (BI), calculus criteria (CC), and plaque index (PI) were measured and scored (as per Table 1) on Day 0 for baseline data. On the same day the drug-loaded periodontal inserts were placed at the selected sites of the treatment group. Patients were called for follow-up clinical evaluations on Day 7, 14, 21, and 28.

TABLE 1 Scoring of clinical parameters

Scale	Gingival index	Bleeding index	Plaque index	Calculus index
0	Normal gingival	No bleeding within 30 sec of probing	Absence of dental plaque	Absence of calculus
1	Mild inflammation, slight color change, and edema	Bleeding within few seconds of probing	Supragingival calculus extends only slightly less than 1/3rd of gingival half of facial or lingual surface of the tooth	Supragingival calculus extending only slightly below the free gingival margin (not more than 1 mm)
2	Moderate inflammation, marked redness, edema, glazing, bleeding on probing	Immediate bleeding on probing	Dental plaque covering more than 1/3rd but less than 2/3rd of the gingival half of the facial or lingual surface of the tooth	Moderate amount of supragingival and subgingival calculus
3	Severe inflammation, marked redness, edema, spontaneous bleeding	Bleeding along gingival sulcus with the slightest touch	Dental plaque covering 2/3rd or more of the gingival half of the facial or gingival surface of the tooth.	Abundance of supragingival and subgingival calculus

Serum Evaluation of NZ by HPLC Method

Six male volunteers in the age group of 23–28 years (26.2 ± 1.76 years) and weighing 50–75 Kg (62. 84 ± 3.55) were enrolled for the study. They were briefed about the study protocol, its objectives, and the risks involved. Full-mouth scaling was carried out on all the 6 volunteers, and periodontal sites with pocket depth equal to or greater than 6 mm were identified and noted against the respective volunteer. All the volunteers were provided similar diet and fluid intake during 24 hr of the study period. Two inserts (RN1 – total dose 1 mg/volunteer) were placed into identified periodontal pockets after obtaining blank blood samples from the volunteers. Blood was further sampled (5.0 ml from an arm vein) at 1, 6, 12, and 24 hr after device insertion. Serum was separated by centrifugation (3200 rpm for 30 min) and stored at -30°C until analysis.

The method of Valencia et al. (1984), was used for estimation of NZ. Briefly, 1 ml of serum was transferred to glass tubes, followed by an equal volume of 1M ammonium phosphate buffer pH 5.5; the sample was mixed and extracted thrice with 5 ml ethyl acetate (HPLC grade), saturated with water. The ethyl acetate extracts were combined in a glass tube over anhydrous sodium sulphate. The anhydrous extract was then transferred into another glass tube for evaporation of the solvent under nitrogen. The residue was reconstituted in 300 μ l of filtered 14% acetonitrile (HPLC grade) -86% 0.1 M sodium acetate buffer pH 5.0. Aliquots of 50 μ l were injected into the HPLC for analysis. The lower limit of detection was defined as a peak with height twice the baseline amplitutde with the recorder set at 0.05 units. Under this condition, the lower limit detectability was 12 ng/ml of serum for NZ.

HPLC was performed with a model LKB Broma (LKB Produkter, AB, Broma, Sweden) equipped with LKB Broma 2150 HPLC pumps, LKB Broma 2152 HPLC controller, and LKB Broma 2238 uvicord SII ultraviolet detector, linked to LKB Broma 2210 two-channel recorder. A LKB Broma 2134-210 10 μ analytical C-18 column (ultropac column with Lichrosorb, RP 18, 10 μ M, 4 × 250 mm) was used for analysis. The mobile phase was 14% acetonitrile–86% 0.1M sodium acetate buffer pH 5.0. The flow rate was maintained at 1.0 ml/hr at ambient temperature. The column effluent was scanned at 365 nm using lamp 2. The identity of NZ peak at retention time (t_R = 26 in) for a flow rate of 1.0 ml/hr was confirmed using a known authentic standard of NZ spiked in blank serum sample.

Statistical Evaluation

Intragroup comparison (longitudinal) between baseline data and subsequent data was carried out by Wilcoxon's signed rank test, in case of nonparametric data. The in vitro data were analyzed using student's t-test. α < .05 was considered to be significant.

RESULTS AND DISCUSSION

The physicochemical properties of the prepared inserts are shown in Table 3. The addition of plasticizer was necessary to obtain drug-loaded films with sufficient stability and to allow subdivision of the films into inserts of uniform dimensions. For this reason, drug release studies from inserts without plasticizer was not undertaken. Weight, thickness, and drug content varied within ± 10% for the prepared inserts.

TABLE 3 Physiochemical characteristics of the prepared inserts

Batch code	Weight (mg ± SD)	Drug content uniformity (mg ± SD)	Thickness (mm ± SD)
RN1	2.0 ± 0.10	0.54 ± 0.04	0.21 ± 0.02
RN2	6.12 ± 0.09	0.54 ± 0.06	0.44 ± 0.04
RN3	8.40 ± 0.61	0.54 ± 0.05	0.64 ± 0.04
RN4	6.83 ± 0.07	1.02 ± 0.08	0.44 ± 0.04
RN5	7.33 ± 0.47	0.51 ± 0.04	0.62 ± 0.05
RN6	7.40 ± 0.36	0.50 ± 0.08	0.39 ± 0.02
RN7	8.27 ± 0.40	0.52 ± 0.05	0.63 ± 0.07
RN8	2.0 ± 0.27	0.51 ± 0.05	0.22 ± 0.02
RN9	6.40 ± 0.55	0.49 ± 0.04	0.48 ± 0.02
RN10	7.53 ± 0.49	0.52 ± 0.04	0.68 ± 0.03
RN11	6.47 ± 0.60	0.99 ± 0.08	0.50 ± 0.03

Drug Release Studies

Inserts containing 7%w/w of NZ (RN3) released NZ at the rate of 0.8 μg/hr until day 6. Incorporation of 3 parts of RG (RN5) decreased the initial rate by half, to 0.4 μ g/hr. As evident from Figure 1, RN3 exhibited a spurt in release around the 10th day, to 1.25 μ g/hr, depleting ∼90% of its drug load in 22 days. RN5 released only 54.1% of its drug load by that time. Kinetic analysis gave an n value of ∼1.0 indicating zero-order release rate and mean dissolution time of 14 days.

FIG. 1 Effect of combination of Resomer RG in RH inserts on niridazole release.

The RG-NZ inserts showed an increase in release rate from 2.02 to 4.05 μ g/hr with an increase in drug loading whereas with the RH-NZ inserts the release rate remained unaffected with drug loading (Figure 2).

FIG. 2 Effect of drug loading on niridazole release from Resomer (RH and RG) inserts.

As shown in the Figure 3, an increase in the concentration of either RH or RG in the respective inserts resulted in a decrease in the cumulative percent drug release. The rate of release was generally slower from RG inserts in comparison to the corresponding RH inserts. This could be due to the presence of relatively lower glycolic acid content in RG inserts.

FIG. 3 Effect of Resomer RH and RG concentration on niridazole release.

Weight variation study was carried out in RG-NZ and RH-NZ inserts to determine their water uptake and the consequent degradation. RG inserts (drug-loaded and placebo) showed very little change from their initial weight during 25 days of the in vitro study while the RH inserts gained weight until Day 10 followed by a sudden decrease in weight as evident from Figure 4. This drop could be attributed to the presence of lower proportions of glycolic acid in RG. It has been shown earlier that the higher the content of glycolic acid in the polymer, the faster is its hydrolysis and consequently rapid water uptake and erosion evidenced by decrease in weight (Vishwanathan et al. 2001; Reed and Gilding 1981; Guinchedi et al. 1994). The decrease in weight around Day 10 coincided with the increase in percent release of the drug.

FIG. 4 Water uptake of niridazole-loaded Resomer RH and RG and placebo inserts.

The release of NZ from the prepared inserts appeared to be triphasic, as was previously observed with PLGA polymeric implantable systems (Furr and Hutchison 1991; Sanders et al. 1994; Bharadwaj et al. 1997). An early rapid release of NZ presumably due to dissolution and diffusion from the surface of the insert, followed by the second phase in which the hydrated polymer underwent hydrolysis and became more soluble in the release medium, was observed. The third phase represented the onset of erosion during which the polymer formed oligomers. The proposed mechanism for Resomer® degradation is random chain scission due to hydrolytic cleavage of the ester bonds in the polymeric backbone (Reed et al. 1981; Fukuzaki et al. 1991). Such devices degrade heterogeneously, the degradation of the inner layers being faster than the surface because of diffusion phenomena involving ester bond cleavage and leaching of water soluble fragment (Grizzi et al. 1995). The first event, which is likely to take place after immersion of the insert in an aqueous medium, was always water uptake followed by homogenous hydrolysis of the ester bonds. At the second stage heterogeneous degradation has been observed, which coincided with a decrease in weight around the 10th day.

Clinical Evaluation

Batch RN1 having the least RH concentration released its entire drug load within 24 days. Further, the device also was completely degraded within this period. Hence it was taken up for the pilot-scale clinical trial. The insert was found to be safe, as no NZ was detected in the serum of the volunteers. The results of the clinical studies were that all the groups showed improvement in the clinical indices from the baseline values. As shown in Table 4, the insert showed the greatest decrease in bleeding index scores, and the average pocket depth was significantly reduced (α = 0.05) at 28 days (from 6.34 ± 1.86 to 5.94 ± 0.28 mm).

TABLE 4 Pilot-scale trial of niridazole insert

	Scores
	Treatment group						Control group
Clinical parameter	Pr-T	0	7 days	14 days	21 days	28 days	Pr-T	0	7 days	14 days	21 days	28 days
Calculus criteria	1.28 ± 0.35	Baseline	–0.16 ± 0.18	–0.38 ± 0.22	–0.47 ± 0.19	–0.81 ± 0.24	1.29 ± 0.27	Baseline	–0.21 ± 0.07	–0.21 ± 0.09	–0.28 ± 0.09	–0.37 ± 0.11
Plaque index	0.74 ± 0.42	Baseline	–0.81 ± 0.32	–0.72 ± 0.35	–0.85 ± 0.47	–0.88 ± 0.52	0.92 ± 0.57	Baseline	–0.72 ± 0.18	–0.86 ± 0.25	–0.91 ± 0.38	–0.92 ± 0.41
Gingival index	1.08 ± 0.61	Baseline	–0.63 ± 0.48	–0.82 ± 0.36	–0.93 ± 0.37	–1.09 ± 0.42	1.25 ± 0.56	Baseline	–0.72 ± 0.47	–0.81 ± 0.51	–0.92 ± 0.38	–0.98 ± 0.71
Bleeding index	1.86 ± 0.97	Baseline	–0.54 ± 0.24	–0.80 ± 0.32	–0.78 ± 0.37	–0.98 ± 0.23	1.98 ± 0.69	Baseline	–0.35 ± 0.21	–0.57 ± 0.18	–0.62 ± 0.16	–0.68 ± 0.17

*Pr-T = Pretreatment scores.

CONCLUSION

The Resomer-based delivery system showed a triphasic release pattern in vitro. Inclusion of RG in RH inserts retarded the drug release. Even though the number of patients in this study was small, the study was of shorter duration, and the rate of degradation of the inserts in the periodontal pocket was not monitored, the clinical evaluation leaves no doubt whatsoever to the effectiveness of the treatment. Additionally, the undetectable levels of NZ in human volunteers in our study show that no dose-related toxicity upon treatment with intrapocket NZ device will be encountered. Hence in our view, the present study could be viewed as a start to the search for a new clinical avenue for NZ, the hitherto orphan drug, NZ. However, long-term clinical trials, comparative studies with other modes of treatment, and the probability of development of resistant strains need to be addressed.

The financial support of the University Grants Commission, Government of India, is gratefully acknowledged.